The division of protein mass spectrometry into the dichotomy of Top Down and Bottom Up has emerged over the past decade. For many of these years Top Down has been seen as a specialized form of mass spectrometry (MS) that is ill-suited for high throughput analysis. The content of this A1-submission for renewal will help to change this perception. All major aspects of Top Down have improved over the last four years, including; front end separations, instrumentation and data analysis. Recent advances have allowed us to detect 1000 proteins and over 4000 isoforms from ~20 milion human cancer cells in 8 runs over a period of a few months. We propose further innovations to refine a sample preparation platform that has evolved from a low-resolution 2-dimensional separation into a 3-dimensional approach over the last funding period. We now propose a combination of solution isoelectric focusing (sIEF) and gel eluted liquid fraction entrapment electrophoresis (GELFrEE) prior to nanocapillary LC-MS/MS using a next-generation Orbitrap (Aim #1). Based on preliminary data obtained through an ongoing collaboration with the group of Alexander Makarov in Bremen, we project a major expansion in the number of labs capable of performing Top Down Mass Spectrometry on complex mixtures. We will continue with an engineering ethos for technology development to define the Human Proteome, and provide ample preliminary data where proteins with multiple PTMs are found to change in cancer cells undergoing the phenomenon of senescence (Aim #2). Unlike apoptosis, senescence occurs when cells irreversibly stop dividing but remain alive. In comparison to apoptosis, the study of senescence is a relatively new field that has become popular in recent years for its strong connection to aging and its potential role in the treatment of cancer. In response to prior review, we have prepared a new Aim 3 and combined our work on cellular-senescence with an expanded set of preliminary data on integral membrane proteins from mitochondria with up to 12 transmembrane domains. Overall, we describe a maturation of the technology where observations made using Top Down (such as the strong up regulation of methylations on a HMG type protein in senescence) can generate hypothesis-driven work on 'high value' observations. Given the growing interest in Top Down Mass Spectrometry, we project major advances over the next granting period for discovery type biological and biomedical research using whole protein mass spectrometry.